A fundamental process in the development of a functional nervous system is the accurate connection of neurons with the target tissues they control. The overall objective of this project has been to identify molecules important in guiding the outgrowth of axons from embryonic neurons. A bioassay system has been developed which tests the ability of axons from embryonic mouse or rat neurons to extend on modified forms of the pathway which they normally use in vivo. This has been possible by using an explant culture system that reproduces the patterned outgrowth of submandibular ganglion axons over the salivary gland epithelium. Monoclonal antibodies are being generated using a novel immunological technique that biases the immune response towards highly conserved epitopes such as the functional sites of molecules. Initially, the primary focus of the study was to raise antibodies against substrates and to use these to identify the molecules involved. This is still the case; however, a new approach has also been undertaken to identify the complimentary molecules present in axonal membranes that mediate recognition of substrate-associated molecules. The specific objectives for the final year of support will be two-fold: 1) To continue to refine the immunization technique to produce monoclonal antibodies specific for the epithelial basement membrane upon which submandibular ganglion neurons travel; and to screen these antibodies by using the in vitro bioassay to test for the ability of these antibodies to block axonal outgrowth; 2) To isolate receptors in the neuronal membrane that recognize substrate molecules such as laminin, a potent inducer of neurite outgrowth, by affinity purification. The affinity purified material will be used to raise antibodies. If such molecules actually mediate outgrowth then antibodies directed against their functional sites should interfere with outgrowth. These antibodies will provide powerful tools for the further study of the regulation of axonal outgrowth during development and regeneration. They will be especially important tools not only in further understanding how these processes may go awry to produce birth defects and neurological malfunction but also in defining conditions necessary for the recovery of function after trauma to the nervous system.